Discussion
While COVID-19 disease presents primarily with respiratory symptoms, for many patients including children, it is a systemic disease with a wide range of effects on many organs (39-41). In this report, we show preliminary evidence for association of caspase molecules that play role in cell death and immunity, not only in the acute phase but also late stages of COVID19 (42). The changes are seen in multiple caspase molecules, and a number of different circulating blood cells, a finding that will further lead to exploration of the systemic nature of this disease.
Caspase-1 has been proposed to play role in the pathophysiology of COVID-19 (43). In addition to leading to a lytic form of cell death called pyroptosis, caspase-1 induces the formation of biologically active IL-18 and IL-1b (26, 44). IL-18 induces an IFN-γ response, while IL-1β induces neutrophil influx and activation, T and B-cell activation, cytokine and antibody production, and promotes Th17 differentiation (45-48). Elevated levels of IL-18, IL-1β, and other proinflammatory cytokines were observed from the lungs and sera of COVID-19 patients (49). Although activation of the inflammasome enhances immunity against pathogens, the accompanying danger and inflammatory signals originating from pyroptosing immune system cells (e.g., T cell and macrophage/dendritic cells) can be damaging to the host in several ways. First, it will result in immune cell lymphopenia, such as that observed with T cells, a pathognomonic feature for SARS-CoV-2, creating an adaptive immune defect. Second, the host will have difficulty controlling the inflammation created in the setting of this immune deficiency as the “danger signals” would also be originating from dying immune system cells (50-52). The end result is likely a self-damaging shut down of the immune system, resulting in acute virus-induced immune deficiency (AVID). Preventing the pyroptotic lymphocyte death by using caspase inhibitors may lead to better success rather than inhibition of the inflammatory response from the cell death itself. The failure of cytokine targeted therapies could be due to that adaptive immune dysfunction due to AVID weighing more heavily than an inflammatory response in disease progression (18).
In our active T-cell caspase-1 assay, we analyzed the sensitivity to nigericin stimulation. This provided further information on the cell surface pannexin-1 expression, which is upregulated by cellular caspases and can play role in disease pathogenesis. Also, cell surface expression level of pannexin-1 can vary between healthy controls, which can explain the differences in response to nigericin. We also found that EMR is effective in reducing active caspase-1 T cells from COVID-19 patients, while VX765 failed to significantly do so. VX765 is a prodrug that needs hydrolyzation to form into its active form and is a reversible inhibitor of caspase-1, as opposed to EMR which is an irreversible inhibitor. Furthermore, EMR is transported into cells via active transport with cell membrane channels, whereas VX765 is internalized by passive diffusion. All these factors may explain the differences we see between these two caspase inhibitors. Further studies are needed to explain the differences in inhibition between the two molecules, particularly in the context of SARS-CoV-2 infection.
The changes in caspase expression are not only limited to T cells, as we show changes in caspase-3 in RBCs and caspase-5 in neutrophils. Caspase activation has been shown to induce changes in the RBC morphology (53-55), which can explain the contamination of the PBMC layer during cell separation as a result of a reduction in their density. Furthermore, their overexpression of caspase 3/7 can subsequently contribute to the formation or advancement of inflammatory microvascular thrombi, which is prominently found in the lung, kidney, and heart of patients with COVID-19 (56, 57). Although viral illnesses typically will impact the function or the life-cycle of lymphocytes, presenting with either lymphocytosis, such as in with CMV, influenza, varicella, or more rarely, lymphopenia, as in H5N1, H1N1, HIV, the finding of neutrophilia in the setting of moderate to severe COVID-19 has been a common, but intriguing finding (58). In the absence of significant overexpression of apoptotic caspases, the increase in the inflammatory caspase-5 in neutrophils may play a part in the neutrophilia observed with COVID-19. Furthermore, the production of IL-10 by neutrophils with increase caspase activity, can further suppress the proliferation of T lymphocytes, hence contributing to the adaptive immune deficiency.
Caspase molecules have been studied extensively in many forms of inflammatory conditions, such as obesity, diabetes and nonalcoholic steatohepatisis (NASH) (59-61). Caspase-1-dependent inflammasome activation has been shown to have a crucial function in the establishment of diabetic nephropathy (62). In an animal model of hypertension apoptosis of myocardial cells were demonstrated, and the apoptosis becomes more serious with the constantly elevated level and prolonged duration of hypertension. The activity of caspase‑3 was shown to have a close correlation with cardiomyocyte apoptosis (63). Our data showing increased expression of active caspase-1 in T-helper cells of patients with asthma and immune deficiencies correlates with their high-risk classification for severe COVID-19 as provided by the centers for disease control (CDC). Perhaps, the changes in cellular caspases seen in COVID-19 may not only explain the multisystem involvement in this disease but may allow for identification for those at risk for complications, including long haulers, based on caspase expression in blood cells.
Our findings suggest a novel alternate therapeutic approach against COVID-19 through the use of caspase inhibition early on in the course of infection to alleviate or prevent disease progression. As an oral formulation, EMR has been shown to reduce serum markers of apoptosis (caspase-3/7), liver enzymes, function (e.g., reducing ALT, MELD & Child-Pugh scores, INR and total bilirubin) and inflammatory biomarkers (CK-18) in patients w/ hepatitis C virus and NASH (64). Although there was no improvement in liver histology, it is possible that the pathology of this disease has mechanisms that are caspase-independent or with the timing of therapy (65, 66). Although SARS-CoV-2 does not seem to infect immune system cells (with the possible exception of macrophage or dendritic cells), the outcome of T cell depletion in severe forms of the disease seems to be through caspase-1 activation, a mechanism also proposed in HIV (67). A better understanding of the impact of different co-morbid conditions on T cell caspase expression at baseline, before exposure to SARS-CoV-2, may identify those that are at highest risk for developing severe disease. There is a large body of evidence pointing out to an activated inflammasome in a wide variety of disorders that overlap with high-risk conditions for severe COVID-19 (15, 51, 52, 68). Ultimately in vivo clinical data is necessary to test the hypothesis of whether pan-caspase inhibition can prevent inflammasome activation in early onset SARS-CoV-2 patients and subsequent lymphopenia and sequelae development. Furthermore, the pan-caspase inhibitor, EMR has been shown in a bioinformatics computational screen to bind to the COVID-19 receptor ACE2, suggesting a potential block to cell entry (69). In a separate unrelated study, a screen of ~6,070 drugs with a known 28 previous history of use in humans was conducted to identify compounds that inhibit the activity of SARS-CoV-2 main protease Mpro in vitro (70). EMR was shown to be among 50 compounds with activity against Mpro with an overall hit rate <0.75%. Preliminary evidence on this multimodal therapeutic effect of EMR raise a relevant key question that will need to be answered through a randomized clinical trial in the setting of COVID-19 (Figure 6).
An important aspect of our study is the demonstration of caspase-1 expression well past the acute stage of COVID-19, suggesting a role in the convalescent phase or disease sequelae. Such persistent changes can not only be limited to immune system cells but can be seen in tissues such as endothelial cells, which could be a causal impact on multiple organ systems (19, 39, 71). Assessing the sequelae, such as fatigue, dyspnea, cough, joint pain, anosmia, among others (32, 72), in correlation with the changes in caspase molecules in natural history studies are warranted. Sequelae targeting populations where caspase elevations are more common, such as the elderly, and those with other co-morbid conditions, such as heart disease, diabetes, hypertension, provides further evidence for the association of caspases with poor outcomes from COVID-19 (1). Dampening the inflammatory response early in the disease process may be a strategy to prevent sequelae, such as in rheumatic fever, where treating streptococcus early on in the disease through the co-treatment of penicillin and anti-inflammatories can prevent severe disease sequelae.